T lymphocytes undergo two selection processes during development in the thymus that are mediated by engagement of the T cell antigen receptor (TCR), termed negative and positive selection. The former involves the induction of programmed cell death in cells with self-reactive specificities, while the latter refers to the induction of differentiation of immature thymocytes as a consequence of recognition of major histocompatibility complex (MHC)-encoded molecules expressed in the thymus. It is unknown what biochemically distinguishes these events, nor how the TCR ligands and antigen presenting cells involved in these processes differ. This research program aims to answer these questions by studying the biochemical and cellular events involved in positive selection, as well as those that induce programmed cell death, using a unique in vitro model system. A thymoma, designated DPK, has been isolated from TCR transgenic mice and adapted to culture in vitro. These tumor cells express immature T cell markers and the transgene-encoded TCR specific for pigeon cytochrome c and class II MHC molecules. Upon stimulation with pigeon cytochrome c and antigen presenting cells, DPK cells undergo a number of phenotypic changes that mimic the positive selection process observed in vivo. A variant of the DPK cell line has also been isolated that undergoes programmed cell death upon cross- linking of the TCR. Together, these cells offer a model system to study both positive and negative selection. Experiments will characterize the functional changes that accompany the differentiation of DPK cells, as well as the proximal biochemical events of positive selection, including changes in intracellular calcium, protein phosphorylation and phosphatidylinositide hydrolysis. Results will be compared to those elicited following activation of the DPK variant that undergoes programmed cell death. Other experiments address the role of the co- receptor CD4 and its associated tyrosine kinase in these processes. Because DPK cells differentiate in vivo in the thymus in the absence of pigeon cytochrome c, they can also be used as an assay for the characterization of the positive selection antigen presenting cell. Ultimately, this system should aid in the identification of the TCR ligand in the thymus. Finally, expression of another TCR in DPK cells by gene transfection will be tested as a means to study development of class I MHC-restricted T cells. The issues concerning T cell development that are the focus of this proposal relate to the biochemical and cellular mechanisms that select the mature T cell repertoire. Certainly these developmental events are central to understanding many facets of autoimmune disease and MHC linked disease susceptibility. Moreover, this series of experiments concerns the balance between growth, differentiation, and cell death of transformed cells, as it relates to intracellular signaling pathways. Elucidation of the requirements to induce programmed cell death or differentiation, and understanding the stages of T cell maturation susceptible to these opposing signals may hold promise for control of neoplastic growth.